Fraydoon Rastinejad

Research Area: Protein Science and Structural Biology
Technology Exchange: Computational biology, Crystallography, Drug discovery, Protein interaction and Transcript profiling
Scientific Themes: Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology

We study ligand-dependent transcription factors, with a current focus on the human basic Helix Loop Helix-PAS (bHLH-PAS) protein family.   Within this  family, class I and class II members heterodimerize to generate functional transcription factors.  Class I members include three hypoxia-inducible factors (HIF-1α, HIF-2α and HIF-3α), the aryl hydrocarbon receptor (AHR), the aryl hydrocarbon receptor repressor (AHRR), four neuronal PAS proteins (NPAS1, NPAS2, NPAS3, NPAS4), two single-minded proteins (SIM1, SIM2), and the clock circadian regulator (CLOCK).  Class II members include the aryl hydrocarbon receptor nuclear translocator (ARNT, also called HIF-1β), ARNT2, brain and muscle ARNT-like protein 1 (BMAL1, also called ARNTL), and BMAL2 (ARNTL2).

A unifying feature of this family is their reliance on PAS domains, whose name derives from the  proteins Period, ARNT, and SIM.  PAS domains evolved for detecting signals in bacteria, archaea and eukaryotic organisms, but in most cases the signals for bHLH-PAS proteins remain unknown.  Further unifying members of this family are their common polypeptide arrangements, where tandem PAS domains (known as PAS-A and PAS-B) lie adjacent to a conserved bHLH DNA-binding domain, and are followed by a variable transactivation domain.

We have recently reported a series of crystal structures showing the bHLH-PAS proteins comprise a distinct family of ligand-binding transcription factors in mammals.  We observed that each of their PAS domains harbors a ligand-binding pocket.   In bHLH-PAS proteins, the PAS domains present an empty pocket at their center. The volumes and the amino-acids lining these pockets are unique for each member of the family, suggesting distinct endogenous ligands should exist.  The functional heterodimers formed in this family bring together four distinct ligand-binding pockets, twice the number in nuclear receptor heterodimers.

Our research program emphasizes ligand discovery for this family, taking into account their individual PAS-A and PAS-B domains.  We are biochemically preparing each individual PAS domain in multi-milligram amounts, and relying on innovative biochemical screening strategies that identify chemical ligands.  To generate a coherent understanding of ligand-dependent actions in this family, we are attempting to link the discovery of chemical ligands to the genomic signatures of each family member. Furthermore, we are interested in addressing mechanistic questions about ligand-modulation in this family.  For this purpose,we are applying structural analyses to study the stereochemical basis for ligand binding and ligand actions through induced conformational changes in these proteins. 

Name Department Institution Country
Peter Ratcliffe FRS Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Wilkinson IVL, Perkins KJ, Dugdale H, Moir L, Vuorinen A, Chatzopoulou M, Squire SE, Monecke S, Lomow A, Geese M et al. 2019. Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a Molecular Target of the Utrophin Modulator Ezutromid. Angew Chem Int Ed Engl, | Show Abstract | Read more

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first-in-class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent KD of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.

Wu D, Su X, Lu J, Li S, Hood BL, Vasile S, Potluri N, Diao X, Kim Y, Khorasanizadeh S, Rastinejad F. 2019. Bidirectional modulation of HIF-2 activity through chemical ligands. Nat Chem Biol, 15 (4), pp. 367-376. | Show Abstract | Read more

Hypoxia-inducible factor-2 (HIF-2) is a heterodimeric transcription factor formed through dimerization between an oxygen-sensitive HIF-2α subunit and its obligate partner subunit ARNT. Enhanced HIF-2 activity drives some cancers, whereas reduced activity causes anemia in chronic kidney disease. Therefore, modulation of HIF-2 activity via direct-binding ligands could provide many new therapeutic benefits. Here, we explored HIF-2α chemical ligands using combined crystallographic, biophysical, and cell-based functional studies. We found chemically unrelated antagonists to employ the same mechanism of action. Their binding displaced residue M252 from inside the HIF-2α PAS-B pocket toward the ARNT subunit to weaken heterodimerization. We also identified first-in-class HIF-2α agonists and found that they significantly displaced pocket residue Y281. Its dramatic side chain movement increases heterodimerization stability and transcriptional activity. Our findings show that despite binding to the same HIF-2α PAS-B pocket, ligands can manifest as inhibitors versus activators by mobilizing different pocket residues to allosterically alter HIF-2α-ARNT heterodimerization.

Daniel B, Nagy G, Czimmerer Z, Horvath A, Hammers DW, Cuaranta-Monroy I, Poliska S, Tzerpos P, Kolostyak Z, Hays TT et al. 2018. The Nuclear Receptor PPARγ Controls Progressive Macrophage Polarization as a Ligand-Insensitive Epigenomic Ratchet of Transcriptional Memory. Immunity, 49 (4), pp. 615-626.e6. | Show Abstract | Read more

Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.

Chandra V, Wu D, Li S, Potluri N, Kim Y, Rastinejad F. 2017. The quaternary architecture of RARβ-RXRα heterodimer facilitates domain-domain signal transmission. Nat Commun, 8 (1), pp. 868. | Show Abstract | Read more

Assessing the physical connections and allosteric communications in multi-domain nuclear receptor (NR) polypeptides has remained challenging, with few crystal structures available to show their overall structural organizations. Here we report the quaternary architecture of multi-domain retinoic acid receptor β-retinoic X receptor α (RARβ-RXRα) heterodimer bound to DNA, ligands and coactivator peptides, examined through crystallographic, hydrogen-deuterium exchange mass spectrometry, mutagenesis and functional studies. The RARβ ligand-binding domain (LBD) and DNA-binding domain (DBD) are physically connected to foster allosteric signal transmission between them. Direct comparisons among all the multi-domain NRs studied crystallographically to date show significant variations within their quaternary architectures, rather than a common architecture adhering to strict rules. RXR remains flexible and adaptive by maintaining loosely organized domains, while its heterodimerization partners use a surface patch on their LBDs to form domain-domain interactions with DBDs.Nuclear receptors (NR) are multidomain proteins, which makes their crystallization challenging. Here the authors present the crystal structure of the retinoic acid receptor β-retinoic X receptor α (RARβ-RXRα) heterodimer bound to DNA, ligands and coactivator peptides, which shows that NR quaternary architectures are variable.

Smith SH, Jayawickreme C, Rickard DJ, Nicodeme E, Bui T, Simmons C, Coquery CM, Neil J, Pryor WM, Mayhew D et al. 2017. Tapinarof Is a Natural AhR Agonist that Resolves Skin Inflammation in Mice and Humans JOURNAL OF INVESTIGATIVE DERMATOLOGY, 137 (10), pp. 2110-2119. | Read more

Wu D, Su X, Potluri N, Kim Y, Rastinejad F. 2016. NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors. Elife, 5 | Show Abstract | Read more

The neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the wider mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.

Wu D, Potluri N, Lu J, Kim Y, Rastinejad F. 2015. Structural integration in hypoxia-inducible factors. Nature, 524 (7565), pp. 303-308. | Show Abstract | Read more

The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-α and ARNT (also called HIF-1β) subunits. Here we describe crystal structures for each of mouse HIF-2α-ARNT and HIF-1α-ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2α-ARNT and HIF-1α-ARNT, wherein ARNT spirals around the outside of each HIF-α subunit. Five distinct pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-α mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.

Santori FR, Huang P, van de Pavert SA, Douglass EF, Leaver DJ, Haubrich BA, Keber R, Lorbek G, Konijn T, Rosales BN et al. 2015. Identification of natural RORγ ligands that regulate the development of lymphoid cells. Cell Metab, 21 (2), pp. 286-298. | Show Abstract | Read more

Mice deficient in the nuclear hormone receptor RORγt have defective development of thymocytes, lymphoid organs, Th17 cells, and type 3 innate lymphoid cells. RORγt binds to oxysterols derived from cholesterol catabolism, but it is not clear whether these are its natural ligands. Here, we show that sterol lipids are necessary and sufficient to drive RORγt-dependent transcription. We combined overexpression, RNAi, and genetic deletion of metabolic enzymes to study RORγ-dependent transcription. Our results are consistent with the RORγt ligand(s) being a cholesterol biosynthetic intermediate (CBI) downstream of lanosterol and upstream of zymosterol. Analysis of lipids bound to RORγ identified molecules with molecular weights consistent with CBIs. Furthermore, CBIs stabilized the RORγ ligand-binding domain and induced coactivator recruitment. Genetic deletion of metabolic enzymes upstream of the RORγt-ligand(s) affected the development of lymph nodes and Th17 cells. Our data suggest that CBIs play a role in lymphocyte development potentially through regulation of RORγt.